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The Synaptonemal Complex Protein ZYP1 Is Required for
Imposition of Meiotic Crossovers in Barley
W OPEN
Abdellah Barakate,a,1 James D. Higgins,b,1,2 Sebastian Vivera,a Jennifer Stephens,c Ruth M. Perry,b Luke Ramsay,c
Isabelle Colas,c Helena Oakey,a Robbie Waugh,a,c F. Chris H. Franklin,b Susan J. Armstrong,b,3 and Claire Halpina,3,4
a Division of Plant Sciences, College of Life Sciences, University of Dundee at The James Hutton Institute, Invergowrie, Dundee DD2
5DA, United Kingdom
b School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom
c The James Hutton Institute, Invergowrie, Dundee DD2 5DA, United Kingdom
ORCID IDs: 0000-0001-6027-8678 (J.D.H.); 0000-0001-6980-9906 (I.C.); 0000-0002-1808-8130 (C.H.)
In many cereal crops, meiotic crossovers predominantly occur toward the ends of chromosomes and 30 to 50% of genes rarely
recombine. This limits the exploitation of genetic variation by plant breeding. Previous reports demonstrate that chiasma
frequency can be manipulated in plants by depletion of the synaptonemal complex protein ZIPPER1 (ZYP1) but conflict as to the
direction of change, with fewer chiasmata reported in Arabidopsis thaliana and more crossovers reported for rice (Oryza sativa).
Here, we use RNA interference (RNAi) to reduce the amount of ZYP1 in barley (Hordeum vulgare) to only 2 to 17% of normal
zygotene levels. In the ZYP1RNAi lines, fewer than half of the chromosome pairs formed bivalents at metaphase and many
univalents were observed, leading to chromosome nondisjunction and semisterility. The number of chiasmata per cell was
reduced from 14 in control plants to three to four in the ZYP1-depleted lines, although the localization of residual chiasmata was
not affected. DNA double-strand break formation appeared normal, but the recombination pathway was defective at later
stages. A meiotic time course revealed a 12-h delay in prophase I progression to the first labeled tetrads. Barley ZYP1 appears
to function similarly to ZIP1/ZYP1 in yeast and Arabidopsis, with an opposite effect on crossover number to ZEP1 in rice, another
member of the Poaceae.
INTRODUCTION
Meiosis consists of two successive nuclear divisions, which result
in the production of four haploid daughter cells (Zickler and
Kleckner, 1998; Cnudde and Gerats, 2005). Meiotic division I is
preceded by DNA replication, a process that generates a pair of
sister chromatids from each original chromosome. Physical
connections between homologous chromosome pairs (homologs)
during prophase I ensure their accurate disjunction and reductional segregation to opposite cell poles, and recombination
between the homologs generates genetic variation. The second,
mitotic-like division, results in the equational segregation of the
sister chromatids to form the four haploid gametes. Meiotic
recombination is initiated by the formation of numerous programmed DNA double-strand breaks (DSBs) by the topoisomerase II–related protein SPO11 during early prophase I
(Keeney et al., 1997; Grelon et al., 2001; Hartung et al., 2007). In
1 These
authors contributed equally to this work.
address: School of Biological Sciences, Adrian Building,
University Road, University of Leicester, Leicester LE1 7RH, UK.
3 Senior authors for Birmingham and Dundee, respectively.
4 Address correspondence to [email protected].
The authors responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) are: Susan J.
Armstrong ([email protected]) and Claire Halpin (c.halpin@
dundee.ac.uk).
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2 Current
most species, including plants, only a small proportion (;5%) of
the DSBs result in the formation of genetic crossovers (COs) with
the remainder repaired as noncrossovers (NCOs). Extensive
studies in budding yeast (Saccharomyces cerevisiae) have shown
that the majority of COs are dependent on a set of proteins referred to as ZMMs (the zipper proteins Zip1, Zip2, Zip3, Zip4; the
MutS homologs Msh4 and Msh5; and Mer3 [meiotic recombination 3]) (reviewed in Lynn et al., 2007). ZMM-dependent
COs exhibit interference, a phenomenon whereby a CO occurring
at a site along a chromosome reduces the probability of additional
COs in adjacent chromosomal regions. Thus, COs are spaced far
apart along the chromosome arms. A small percentage of COs
(;15%) arise independently of the ZMMs and are distinct in that
they do not exhibit CO interference. It is proposed that NCOs
arise via synthesis-dependent strand annealing rather than from
double Holliday junctions that are the defining feature of the CO
pathway (Schwacha and Kleckner, 1995; Allers and Lichten, 2001;
Hunter and Kleckner, 2001; McMahill et al., 2007). Studies in the
model plant Arabidopsis thaliana and other plant species, such as
rice (Oryza sativa), maize (Zea mays), and barley (Hordeum
vulgare), suggest that CO formation is very similar to that in
budding yeast despite some differences in detail (Osman et al.,
2011). The nature of the NCO in plants has not yet been determined. In fission yeast, the helicase Fml1 (Fanconi anemia
complementation group M-like protein) has been shown to direct
NCO formation (Lorenz et al., 2012). In Arabidopsis, the FANCM
helicase protein plays a major role in limiting meiotic CO, suggesting a potential role in NCO formation (Crismani et al., 2012;
Knoll et al., 2012). Direct analysis of NCOs and their distribution
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has been difficult as they are hard to detect; however, recent
sequencing approaches are beginning to yield insights (e.g.,
Drouaud et al., 2013; Wijnker et al., 2013).
Evidence from budding yeast indicates that the fate of individual DSBs to proceed via the CO or NCO route is designated
prior to the establishment of a stable single end invasion intermediate. Imposition of this fate is dependent on the ZMMs and
coordinated with the formation of the synaptonemal complex
(SC), a tripartite proteinaceous structure that holds the homologs
in close apposition during pachytene (Heyting, 1996; Zickler and
Kleckner, 1999). The SC is comprised of two lateral elements
derived from the axial elements that are established during leptotene and that organize each pair of sister chromatids into linear
looped arrays conjoined at the loop bases (Zickler and Kleckner,
1999). During zygotene, the Zip1 protein forms a transverse filament that polymerizes along the aligned lateral elements to form
a zipper-like structure. In budding yeast, SC formation is initiated
at discrete synapsis initiation centers that are coincident with
CO-designated ZMM recombination intermediates (Börner et al.,
2004). Studies in Arabidopsis have also revealed that SC initiation
sites colocalize with ZMM proteins, such as MSH4 (Higgins et al.,
2004). However, the number of synapsis initiation centers appears
to be greater than the eventual number of COs, suggesting an
additional level of control. In budding yeast, zip1 mutants exhibit
defects in SC formation and recombination. The nature of the
recombination defect is temperature dependent. At 33°C, DSBs
and NCOs occur as normal, but interference-dependent COs are
absent due to a failure of progression from DSBs to stable strand
end invasion intermediates. At 23°C, zip1 mutants display a different phenotype; strand end invasion and double Holliday junction intermediates are formed with a delay relative to the wild type,
but there is an overall reduction in COs and a corresponding increase in NCOs. Thus, it appears that CO designation occurs, but
this is not maintained, and CO fate specification is ultimately lost
(Börner et al., 2004).
Previous studies in Arabidopsis, rice, maize, and wheat (Triticum aestivum) have identified plant Zip1 orthologs (Higgins et al.,
2005; Wang et al., 2010; Golubovskaya et al., 2011; Khoo et al.,
2012). Arabidopsis contains two partially redundant proteins
ZYP1a and ZYP1b (collectively ZYP1), whereas rice possesses
a single protein referred to as ZEP1. Rice ZEP1 exhibits 42%
identity with ZYP1b and 41% identity with ZYP1a. The plant
proteins exhibit little primary sequence identity with Zip1 and
transverse filament proteins from other species, such as c(3)G in
Drosophila melanogaster and Sypc1 in mouse (Page and Hawley,
2001; de Vries et al., 2005). Despite this lack of primary sequence
homology, the plant proteins show structural similarity to the
transverse filament proteins from other species. All comprise
coiled-coil proteins with globular domains at their N and C termini
(Suzuki, 1989). This is in keeping with the close ultrastructural
similarity of the SC observed by electron microscopy in a wide
variety of species (Zickler and Kleckner, 1999). Arabidopsis and
rice lines lacking these transverse filament proteins through mutation or RNA interference (RNAi) knockdown fail to form SCs and
exhibit defects in CO control (Higgins et al., 2005; Wang et al.,
2010). In the case of Arabidopsis, a reduction in chiasma frequency to around 70% is observed. This is accompanied by
extensive multivalent formation due to ectopic recombination
possibly between chromosomal regions that have undergone
segmental duplication during the evolution of this species (Higgins
et al., 2005). Unexpectedly, and in contrast with Arabidopsis and
other species, zep1 mutants in rice are reported to exhibit an increase in chiasma frequency, with 3 to 4 times the number of COs
observed on the short arm of chromosome 11 in the mutant
compared with the wild type (Wang et al., 2010).
In members of the Poaceae family, such as barley, wheat, rye
(Secale cereale), and oat (Avena sativa), meiotic COs predominantly occur in the distal regions of the chromosomes
(Sandhu and Gill, 2002; Mayer et al., 2011). Studies indicate that
this pattern is strongly influenced by the timing of recombination
initiation (Higgins et al., 2012). A consequence of this is that interstitial chromosomal regions encoding an estimated 30 to 50%
of the gene complement rarely recombine. This is a significant
problem for plant breeding programs as it limits access to the
available genetic variation and hampers both gene isolation and
the introgression of new genetic traits. Since rice is a member of
the Poaceae, the observation that the loss of ZEP1 led to an increase in recombination suggested a potential route to increase
CO formation in the nonrecombining chromosomal regions in
other cultivated members of the family, such as barley.
In this study, we used RNAi to knock down expression of
ZYP1 that encodes the barley ZEP1 homolog. Quantitative realtime PCR and immunocytochemistry confirm that transcript and
protein expression has been reduced to very low levels in the
transgenic barley lines; as a result, they fail to form a SC at
pachytene. Surprisingly, these lines exhibit a recombination
defect that contrasts starkly with that reported for rice. Rather
than an increase in COs, downregulation of ZYP1 results in
a dramatic reduction in CO formation that is reminiscent of that
observed in zip1 mutants of budding yeast and Arabidopsis.
RESULTS
Production of ZYP1 Knockdown Barley Lines
We previously reported cloning of the full-length barley ZYP1
cDNA (Higgins et al., 2012). Alignment of the predicted protein
sequence with other plant full-length ZYP1 sequences and assembly of a phylogenetic tree (Figure 1A; Supplemental Data Set
1) reveal clear separation of the monocot and dicot sequences
and confirm that the barley gene is closely related to the functionally characterized rice ZEP1 (Wang et al., 2010). ZYP1 expression levels determined in different organs of barley cv Golden
Promise using quantitative real-time PCR (qPCR) confirmed that
the transcript was highly abundant in young meiotic spikes
(0.5- to 1.5-mm anthers) (Figure 1B).
An RNAi construct composed of an 800-bp fragment of the
ZYP1 cDNA in the Gateway-based hairpin vector pBRACT207 was
used to generate 43 independent ZYP1RNAi lines in barley cv
Golden Promise. Transgene integration was confirmed by PCR of
the hygromycin resistance gene from plant genomic DNA. At maturity, the majority of the T0 plants showed reduced fertility compared with empty vector control plants, and some did not grow to
normal height. Only plants with normal vegetative phenotypes were
used for subsequent studies. The number of loci where transgenes
ZYP1 and Meiotic Crossover in Barley
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screened for reduced fertility as an indicator of a possible meiotic
defect. Seed numbers per spike were scored for plants of each of
the five selected lines and the empty vector control (Figure 3;
Supplemental Figure 2). All ZYP1RNAi transgenic plants showed
a strong reduction in the number of seeds per spike compared
with the control lines.
SC Formation Is Severely Affected by ZYP1 Knockdown
Figure 1. ZYP1 Expression and Phylogenetic Tree.
(A) ZYP1 phylogenetic tree using full-length amino acid sequences from
plants and D. melanogaster as the out-group. Numbers indicate statistical support, provided as posterior probabilities. The scale bar at the
bottom gives the number of expected substitutions per site.
(B) ZYP1 mRNA levels in different organs of barley cv Golden Promise
were monitored using qPCR. Three samples each were collected from
four plants. Error bars represent SD (n = 12).
had integrated was determined by the segregation of hygromycin
resistance in germinating T1 seeds (Supplemental Table 1). In addition, a cytological analysis of 49,6-diamidino-2-phenylindole
(DAPI)–stained chromosome spreads from meiocytes of T0 plants
at metaphase I was conducted to determine whether there were
any chromosome abnormalities. Based on these analyses, five
lines that appeared to have a single locus where a transgene (or
transgenes) had inserted and no obvious chromosomal aberrations
were selected for further study and production of homozygotes.
Lines selected were ZYP1RNAi15, ZYP1RNAi24, ZYP1RNAi30,
ZYP1RNAi35, and ZYP1RNAi43 plus two empty vector control lines.
The presence of a single transgene locus in these lines was confirmed by fluorescence in situ hybridization (FISH) on meiotic prophase I chromosome spreads using the pBRACT empty vector as
a probe. ZYP1RNAi lines 15, 24, 30, 35, and 43 appeared to contain
a single homozygous transgene insertion locus, whereas the empty
vector line contained two transgene insertion loci (Supplemental
Figure 1), and a nontransformed plant produced no signal.
Substantial reductions of ZYP1 mRNA were evident by qPCR
in the inflorescences of T1 plants of the selected ZYP1RNAi lines
(15, 24, 30, 35, and 43) compared with their segregating azygous
siblings (i.e., the T1 progeny that lost the transgene through
segregation) and empty vector controls (Figure 2). The lines were
ZYP1 forms the transverse filaments of the SC in plants and has
been identified in Arabidopsis, rice (known as ZEP1), maize,
barley, and wheat (Higgins et al., 2005, 2012; Wang et al., 2010;
Golubovskaya et al., 2011; Khoo et al., 2012). ZYP1 is initially
observed in chromosome spread preparations from wild-type
barley meiocytes at leptotene as foci in the subtelomeric regions
(Higgins et al., 2012). These elongate to form stretches concurrent with further foci appearing in the chromosomal interstitial
and proximal regions, and the stretches continue to extend and
coalesce until full synapsis is achieved at pachytene (Higgins
et al., 2012). In this study, the same pattern was observed by
immunolocalization of ZYP1 during prophase I using the Arabidopsis ZYP1 antibody on the empty vector control (Figures 4A to
4E; Supplemental Figure 3A). In the ZYP1RNAi lines, ZYP1 protein
expression was significantly depleted such that none of the cells
analyzed (n = 157) achieved full synapsis; nevertheless, it was
also rare for there to be a complete absence of the protein
(Figures 4F to 4J; Supplemental Figure 3B), consistent with the
qPCR data showing residual transcript levels (Figure 2). The
majority of cells had multiple foci and short stretches of ZYP1
with occasional longer stretches also observed.
To quantify the amount of polymerized ZYP1 for each cell, the
total length of the stretches was measured by tracing chromosomes using the Nikon NIS-Elements software. We have previously
shown that nuclei at the beginning of zygotene have a large envelope volume (i.e., the volume of all of the chromatin, as defined in
Kleckner et al., 2004), which decreases as ZYP1 polymerization
progresses (Higgins et al., 2012). Therefore, to accurately determine
the meiotic stage of cells, it was necessary to take into account
Figure 2. ZYP1 mRNA Level in the Spikes of Five RNAi Lines.
Expression of ZYP1 in meiotic inflorescences of five independent RNAi
T1 lines (ZYP1RNAi15, ZYP1RNAi24, ZYP1RNAi30, ZYP1RNAi35, and
ZYP1RNAi43) relative to control plants (empty vector [EV]) monitored using qPCR. Error bars represent SD (n = 3 to 12, depending on the line).
The ZYP1 mRNA level in each ZYP1RNAi line was significantly lower than
in the empty vector control (P < 0.05). Segregating azygous (AZ) progeny
lacking the transgene were available for three ZYP1RNAi lines (15AZ,
30AZ, and 35AZ) and were included for comparison.
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spread preparations in the transgenic lines. The empty vector
control line was indistinguishable from the wild-type plant. Pachytene nuclei were commonly observed during prophase I, where the
homologs were closely juxtaposed (Figure 5A). This was followed
by metaphase I where seven bivalents aligned, distinguished by 5S
and 45S rDNA FISH probes (Figure 5B). At metaphase II, the seven
pairs of homologs had migrated to the poles and by telophase II,
the sister chromatids had separated to leave four haploid cells
containing seven chromosomes (Figures 5C and 5D). By contrast,
pachytene nuclei were never observed in the ZYP1RNAi lines, although many stages similar to zygotene were found (Figure 5E;
Supplemental Figures 4A, 4E, 4I, and 4M). At metaphase, the
number of bivalents per cell was reduced by over half from 7
(n = 60) in the empty vector control to an average of 3.13 (n = 255) in
the ZYP1RNAi lines, and many univalents were observed (Figure 5F;
Supplemental Figures 4B, 4F, 4J, and 4N). This led to chromosome
nondisjunction at metaphase II and telophase II (Figures 5G and 5H;
Supplemental Figures 4C, 4D, 4G, 4H, 4K, 4L, 4O, and 4P).
Number of Chiasmata per Meiocyte Is Reduced, but
Localization Remains Distal
Figure 3. ZYP1 Suppression Induces Sterility in Barley.
(A) Representative mature spikes of control (empty vector [EV]) and five
different RNAi lines. Compared with the fertile EV control, all ZYP1RNAi
lines are semisterile with only a few seeds.
(B) Seed number distribution per spike of four segregating plants of
ZYP1RNAi43 and an empty vector control plant (EV7).
nucleus size as well as ZYP1 total length. In the empty vector
control, short stretches of ZYP1 were observed in nuclei with very
large nuclear volumes and as these decreased, ZYP1 length increased (Figure 4K). Total ZYP1 length reached its peak at late
zygotene and then decreased to pachytene, in a pattern described
by a polynomial curve. At late zygotene, ZYP1 protein loading is not
complete between the chromosome axes (Figures 4D and 4K), but
as it reaches completion, the length of ZYP1 labeled material
paradoxically reduces (Figure 4K). This is due to the chromosome
axes decreasing in length between late zygotene and pachytene.
Therefore, a polynomial curve was used to fit data points from
total length of ZYP1 stretches in the ZYP1RNAi lines and to
compare with the peak ZYP1 polymerization in the empty vector
(nuclei size = 1000 mm2). At this peak point, total ZYP1 length in
the empty vector was 566 mm compared with 13 to 95 mm in the
ZYP1RNAi lines, a reduction of 83 to 98%. Values in individual
transgenic lines were 50 mm in ZYP1RNAi15 (8% of empty vector),
56 mm in ZYP1RNAi24 (10%), 28 mm in ZYP1RNAi30 (5%), 13 mm in
ZYP1RNAi35 (2%), and 95 mm in ZYP1RNAi43 (17%).
Bivalents Are Reduced, Leading to
Chromosome Nondisjunction
To gain insight into the effect of ZYP1 depletion on chiasma
formation, we performed a cytological analysis on chromosome
There was a significant reduction in the mean number of chiasmata
per cell between the empty vector (14.18 6 0.13, n = 60) and the
ZYP1RNAi lines (3.82 6 0.19, n = 255) (P < 0.001, 314 df ), and this
was consistent across all chromosomes (Supplemental Figure 5).
The reduction of chiasmata varied between lines ranging from
ZYP1RNAi35 with a mean of 1.4 6 0.18 (n = 54) chiasmata per cell to
ZYP1RNAi30 with 6.3 6 0.42 (n = 53). The number of chiasmata per
cell in the empty vector plant differed significantly from a Poisson
distribution [x(19)2 = 107.06; P < 0.001], indicating a nonrandom
underlying process and highlighting the existence of CO control
mechanisms. However, the numerical distribution of residual chiasmata in ZYP1RNAi35 followed a Poisson distribution [x(7)2 = 4.73;
P > 0.10], indicating loss of that control (Figure 6). This observation
is similar to previous studies in zmm mutants in Arabidopsis and
rice and consistent with a CO control defect leading to a loss of CO
assurance (i.e., the obligate establishment of at least one CO per
chromosome pair) (Higgins et al., 2004; Wang et al., 2012). Nevertheless, the distribution of the residual chiasmata in the ZYP1RNAi
lines remained confined to the distal regions (974/975 chiasmata
observed in 255 cells) of the chromosomes similar to that in the
control (785/818 chiasmata observed in 60 cells) and previously
reported in the wild type (Higgins et al., 2012).
DSB Formation Appears Normal but the Recombination
Pathway Is Defective
Although it is not yet possible to directly assay meiotic DSB formation in plants, immunolocalization of recombination proteins can
be used to infer the number of breaks based on the number of foci
observed in early prophase I. Antibodies against gH2AX, the
phosphorylated form of H2AX that marks DSBs, and recombination
pathway proteins including RAD51 and DMC1 have been extensively used for this purpose (Sanchez-Moran et al., 2007; Ferdous
et al., 2012; Higgins et al., 2012). The number of gH2AX foci observed in chromosome spreads from meiocytes at leptotene did
not differ significantly between empty vector controls and ZYP1RNAi
ZYP1 and Meiotic Crossover in Barley
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Figure 4. Quantification of ZYP1 Expression during Meiotic Prophase I.
(A) In the empty vector control, immunolocalization of ZYP1 (green) is first observed as numerous foci throughout the nucleus on the chromosome axes
marked by ASY1 (red).
(B) to (E) During zygotene, short stretches of ZYP1 are observed that elongate until reaching full synapsis at pachytene (E).
(F) to (J) In the ZYP1RNAi lines (numbers shown in top right of image) the majority of nuclei contained a small number of ZYP1 foci and short stretches.
Nuclei were counterstained with DAPI. Bars = 10 mm.
(K) Total ZYP1 lengths for each cell were measured by chromosome tracing and plotted against nuclei size to determine the correct stage. Polynomial
curves were fitted to the data collected from the empty vector control and the numbered ZYP1RNAi lines.
lines (Figures 7A and 7F; Supplemental Figure 6 and Supplemental
Table 2). RAD51 foci counts also did not differ (Figures 7B and 7G;
Supplemental Figure 7 and Supplemental Table 2). This suggests
that DSB formation is unaffected in the ZYP1RNAi lines analyzed in
this study.
The meiosis-specific MutS homolog 4 (MSH4) is essential for
;85% of COs in Arabidopsis (Higgins et al., 2004). In barley,
MSH4 localizes to the emergent chromosome axes during early
leptotene as numerous foci associated with the distal chromosomal regions. As leptotene progresses, MSH4 foci increase to
;400 per nucleus distributed across the length of the mature
chromosome axes, then gradually reduce such that at early
zygotene around 18 to 20 foci remain (Higgins et al., 2004, 2012).
In the empty vector control line, the number of MSH4 foci was
indistinguishable from wild-type plants, accumulating to a maximum of 394 6 6 (n = 10) at mid/late leptotene before reducing in
number. The average number of MSH4 foci at early/mid leptotene
from across the ZYP1RNAi lines was not significantly different to
the control (406 6 18, n = 26) (P = 0.69, 35 df) (Figures 7C and 7H;
Supplemental Figure 8 and Supplemental Table 2). However, by
late leptotene, when ASY1 polymerization was complete, these
had disappeared to leave a number of large MSH4 foci (45 6 3,
n = 29) that were not observed in the wild type or empty vector
control lines (Figures 7D and 7I; Supplemental Figure 9 and
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Figure 5. A Cytological Analysis of Meiotic Chromosomes in ZYP1RNAi.
In the empty vector control, chromosomes are in close alignment during prophase I (A). At metaphase I, bivalents of the seven barley chromosomes, 1H
to 7H, can be distinguished with 5S (green) and 45S (red) ribosomal DNA fluorescence in situ hybridization probes (B). At metaphase II, the homologs
have separated so there are seven chromosomes at each pole (C). At telophase II, the sister chromatids have separated to give four haploid cells each
with seven chromosomes (D). In a representative ZYP1RNAi line (35), chromosomes never become fully aligned during prophase I (E). At metaphase I,
a large number of univalents are observed with occasional bivalents (F). Reduced chiasmata at metaphase I led to chromosome nondisjunction at
metaphase II (G) and telophase II (H). Nuclei were stained with DAPI. Bars = 10 mm.
Supplemental Table 2). They persisted to zygotene when the
short stretches of SC that can still form in the ZYP1RNAi lines are
present but had disappeared by late prophase I.
The MutL homolog 3 (MLH3) is a meiosis-specific protein
required to form normal levels of interference-sensitive COs in
Arabidopsis (Jackson et al., 2006). In barley, it marks late recombination nodules, the future sites of interference-dependent
COs (Phillips et al., 2013). The number of MLH3 foci during
prophase I in the empty vector control line (15 6 2.4, n = 10) was
significantly greater (P < 0.001, 51 df) than the number of pooled
MLH3 foci from the ZYP1RNAi lines (2 6 0.2, n = 42) (Figures 7E
and 7J; Supplemental Figure 10 and Supplemental Table 2).
MLH3 foci numbers ranged from 11 to 18 in the empty vector
control and 0 to 6 in the ZYP1RNAi lines. The numerical distribution of MLH3 foci per meiocyte did not differ significantly from
a Poisson distribution [x(6)2 = 8.13; P > 0.05] consistent with
a breakdown in CO control leading to a random loss of Class I,
interference-dependent COs (Jackson et al., 2006). Collectively,
these data suggest that, while DSBs appear to occur normally in
ZYP1RNAi plants, aspects of the later stages of the recombination
pathway are compromised leading to a reduction in CO formation
but not DSB repair.
Meiotic Progression Is Delayed by ZYP1 Depletion
In Arabidopsis, depletion of the ZYP1 protein perturbs the
meiotic program, leading to a delay during prophase I (Higgins
et al., 2005). Therefore, if completion of chromosome synapsis is
acting as a checkpoint during prophase I, depleting the ZYP1
protein in barley may lead to a delay and alter meiotic progression. To investigate this possibility, we performed a meiotic
time course using bromodeoxyuridine (BrdU) using the empty
vector control and ZYP1RNAi43 plants. In the empty vector
Figure 6. Chiasma Frequency in ZYP1RNAi35 and the Empty Vector
Control Compared with the Predicted Poisson Distribution.
Metaphase I chromosomes stained with DAPI and labeled with 5S and
45S rDNA FISH probes were scored for chiasmata in ZYP1RNAi35 (black)
and the empty vector control (dark gray). The chiasma frequency in the
control does not fit a Poisson distribution (white), whereas chiasmata in
ZYP1RNAi35 do not significantly differ from a Poisson distribution (light
gray).
ZYP1 and Meiotic Crossover in Barley
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Figure 7. Immunolocalization and Quantification of Recombination Protein Loading in ZYP1RNAi30 and the Empty Vector Control.
Markers for early meiotic recombination events, such as gH2AX, RAD51 and MSH4, are indistinguishable between ZYP1RNAi30 ([F] to [H]) and the
empty vector control ([A] to [C]). In ZYP1RNAi30, later events are affected as there is an increased number of large MSH4 foci compared with the control
([I] compared with [D]) and MLH3 foci are reduced ([J] compared with [E]). Numbers represent average number of foci labeled per cell 6 SE (n = 5 to 10).
Full quantification data are shown in Supplemental Table 2, and exemplar images for all lines are in Supplemental Figures 5 to 9. Bars = 10 mm.
control, the first labeled tetrads were identified after 43 h (Figure
8A). One ZYP1RNAi43 plant progressed to first labeled tetrads
after 55 h, an approximate 12-h delay (Figures 8B to 8F), while
cells of four other plants remained in prophase I. Thus, reduction
of ZYP1 expression causes a substantial delay in prophase I
progression.
DISCUSSION
We identified and cloned barley ZYP1 and suppressed its expression to very low levels using a constitutively expressed RNAi
construct to evaluate its role in meiotic CO formation. Our results
suggest an essential role for ZYP1 in ensuring that CO-designated
DSB sites progress to form late recombination intermediates and
full COs. Without ZYP1, CO numbers are dramatically reduced. In
this respect, barley ZYP1 appears to function similarly to ZIP1/
ZYP1 in yeast and Arabidopsis, respectively, and has an opposite
effect on CO number to that reported for its ortholog, ZEP1, in rice,
another member of the Poaceae.
ZYP1 Protein Is Critical for Synapsis and Timely Prophase I
Progression in Barley
Prophase I meiocytes of our ZYP1RNAi transgenic lines had greatly
reduced stretches of SC ranging from 2 to 17% of that of the
empty vector control, consistent with ZYP1 forming the transverse filaments of the SC. Although fertility was dramatically reduced, all transgenic lines produced some seed, and this is likely
due to the fact that occasional meiocytes retained longer tracts of
SC and more residual chiasmata than the average. A minimum
delay of 12 h in prophase I was observed in the ZYP1RNAi43 line,
which was the line with most residual ZYP1 protein. This is
consistent with data from Arabidopsis where a 6- to 20-h delay
was seen in ZYP1 T-DNA mutants or RNAi knockdown plants
(Higgins et al., 2005). Based on studies in budding yeast and
mouse meiotic mutants, it has been proposed that such delays
may be associated with the monitoring of prophase I by a surveillance system that ensures correct regulatory responses to
meiotic defects (Hunt and Hassold, 2002; Börner et al., 2004;
Barchi et al., 2005). The prophase I delay observed in barley in
response to reduced ZYP1, taken in conjunction with the previous
results in Arabidopsis, provides evidence consistent with a prophase I surveillance system in all plants.
ZYP1 Is Required to Implement CO Formation in Barley
The early stages of prophase I appear unaltered in the ZYP1RNAi
lines. This is consistent with data from barley and maize suggesting that a role demonstrated for budding yeast Zip1 in centromere coupling during early meiosis (Tsubouchi and Roeder,
2005) is not conserved in these grasses, where ZYP1 is not detected in centromeres at leptotene (Phillips et al., 2012; Zhang
et al., 2013). Our immunolocalization studies using anti-gH2AX
and anti-RAD51 antibodies suggest that DSB formation and the
initial stages of recombination proceed as normal in the ZYP1RNAi
lines. MSH4 localization also appears to be normal with ;400 foci
up to mid leptotene but then, instead of a gradual transition to
;20 foci in zygotene, there is precocious loss in the ZYP1RNAi
lines such that the number of foci decreases from wild-type levels
at mid leptotene down to ;40 foci at late leptotene. Morphologically these foci appear larger than normal, suggesting that
they correspond to protein accumulations. However, they are
consistent in size and number and are axis associated as they
colocalize with ASY1, suggesting that they are not the protein
polycomplexes that are observed in some meiotic mutants (Chen
et al., 2011). It is tempting to speculate that they may correspond
to sites of recombination intermediates that have somehow
stalled or are progressing more slowly due to the reduced levels
of ZYP1. One possibility is that they mark synapsis initiation sites.
The defect in MSH4 localization is accompanied by a reduction in
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The Plant Cell
Figure 8. Time-Course Determination of the Effect of ZYP1RNAi on the Duration of Meiosis.
BrdU (green) was incorporated into DNA during premeiotic S-phase, and samples were fixed at various time intervals. In the empty vector control, BrdUlabeled tetrads were observed at 43 h (A), whereas the furthest stage reached in ZYP1RNAi43 at 43 h was early prophase (B). This delay in meiosis
progression in ZYP1RNAi43 continued at 47 h (C) and 53 h (D) until labeled metaphase I nuclei were detected at 54 h (E). Labeled tetrads were finally
observed only at 55 h (F) in ZYP1RNAii43. Bars = 10 mm.
the number of MLH3 foci. Biochemical studies using purified
human MSH4/MSH5 indicate that the complex binds and stabilizes proto-Holliday junctions following single-end invasion
(Snowden et al., 2004). As it appears that this function is
conserved in other species including plants (Wang et al., 2012),
the destabilization of MSH4 localization in the ZYP1RNAi lines
would suggest a defect in the formation of late recombination
intermediates.
The meiotic phenotype of the barley ZYP1RNAi lines indicates
that, like Zip1 in budding yeast, ZYP1 is essential for normal
levels of CO formation. A reduction in mean chiasma frequency
to 3.82 per meiocyte was observed for the lines as a whole, ;10
to 44% of the empty vector control. Based on our observations,
we propose that ZYP1 is recruited to early recombination
pathway intermediates. In the ZYP1RNAi lines, CO designation
still occurs, possibly inefficiently, but due to a reduced level of
ZYP1 protein, CO imposition is aberrant. As a result, CO formation is reduced with a proportion of CO-designated intermediates reverting to a non-CO fate or being repaired via some
other route such as intersister chromatid recombination.
In budding yeast, it is proposed that the ZMM proteins ensure
that CO-designated recombination intermediates and structural
components of the meiotic chromosomes are correctly coupled,
thereby allowing normal progression of recombination (Börner
et al., 2004). At designated CO sites, the formation of stable
single-end invasion intermediates is accompanied by SC nucleation to produce a recombination complex. In the absence of
Zip1 or other ZMMs, CO formation is impeded in a temperaturedifferential manner (see Introduction) (Börner et al., 2004). Our
data are consistent with ZYP1 performing a similar role in the
barley recombination pathway.
Residual Chiasmata in the ZYP1RNAi Lines Are
Strongly Localized
The number of chiasmata per nucleus in EV control plants was
significantly more overdistributed around a mean of 14 (i.e., two
per chromosome) than a Poisson distribution would predict.
This nonrandom control was lost in the ZYP1RNAi lines where the
number of residual chiasmata/COs per nucleus fitted a Poisson
distribution around a mean of 1.4 6 0.18 (n = 54), suggesting
a random process. These data clearly indicate that ZYP1 plays
an important role in CO control. However, the position of residual COs in ZYP1RNAi lines was highly regulated with virtually
all occurring in the distal regions of the chromosomes. This is
similar to what is normally found in wild-type barley where interstitial chiasmata are generally rare (Higgins et al., 2012). In the
control lines analyzed here, they accounted for only 0.04% of
the total chiasmata. In the ZYP1RNAi lines, the mean frequency of
chiasmata was 27% of the control, but there was a near complete absence of interstitial chiasmata, a proportionally greater
effect than on distal chiasmata. This may simply reflect the fact
that the initiation of recombination and synapsis in barley occurs
in subtelomeric DNA in advance of interstitial events (Higgins
et al., 2012). When ZYP1 protein is limiting, it may be preferentially recruited to distal regions, further restricting any possible
recruitment to interstitial sites. It is conceivable that other factors
may also influence this. For example, the chromosome bouquet
that forms at early zygotene, clustering telomeres on the nuclear
membrane (Scherthan, 2001), may promote the stability of interhomolog recombination interactions that are close to the telomeric region.
Reduced SC Transverse Filament Protein Produces
Different Effects in Closely Related Grass Species
The proteins that form the transverse element of the SC have
been identified in a number of species; these include Zip1 in
budding yeast, SYCP1 in mouse, C(3)G in D. melanogaster, and
SYP-1/SYP-2 in Caenorhabditis elegans. In all cases, mutation
of the corresponding genes results in a failure to form SC together with a reduction in CO formation (Sym et al., 1993;
Heyting, 1996; Page and Hawley, 2001; MacQueen et al., 2002).
Interestingly, in SYP-1 RNAi C. elegans where sufficient SYP-1
is retained to permit assembly of the SC, a reduction is observed
in the distance over which interference operates, and the number of COs is increased (Libuda et al., 2013). This is likely
a consequence of the distinct way that C. elegans controls CO
designation, which takes place after the assembly of the SC. By
contrast, in budding yeast, CO designation occurs before stable
ZYP1 and Meiotic Crossover in Barley
single end invasions (i.e., before SC formation begins), although
ZYP1 is subsequently required to implement the designated CO
fate (Hunter and Kleckner, 2001; Börner et al., 2004). This seems
to be the case in plants also; in Arabidopsis T-DNA lines lacking
either one of two duplicated ZYP1 genes, there is a modest 10%
reduction in chiasma frequency, whereas Arabidopsis ZYP1RNAi
knockdown lines display a 30% chiasma reduction (Higgins
et al., 2005). Most of the remaining Arabidopsis events involve
ectopic recombination very likely between duplicated regions
located on nonhomologous chromosomes.
Surprisingly, and in contrast with other species, a study in rice
has reported a substantial increase in recombination in mutants of
the ZYP1 ortholog, ZEP1 (Wang et al., 2010), although the CO sites
were not immunologically validated as they had been in the
C. elegans work. Nevertheless, this hyperrecombination phenotype
prompted our current study as it suggested a potential route to
breaking up recombinationally silent regions within the chromosomes of closely related cereals by downregulation of ZYP1/ZEP1.
However, our data clearly demonstrate that in barley, as in all other
species analyzed to date including Arabidopsis, severe ZYP1 depletion dramatically reduces COs. An explanation for this difference
between the closely related barley and rice may lie in the fact that
some of the stronger alleles in the rice work expressed ZEP1
truncated protein (Wang et al., 2010). Moreover, since robust
markers for interference-dependent CO sites were not used in the
rice study, it remains unknown whether these additional COs were
interference dependent and/or interference independent. It is possible that the additional rice COs arose via a pathway that does not
require the posited recombination-promoting function of ZEP1
during the early stages of recombination. Nevertheless, the possibility remains that multiple layers of CO control that make varying
levels of relative contributions in different organisms, as suggested
in reconciliation of the differences between C. elegans and yeast
(Libuda et al., 2013), might also apply between related plant species. In summary, our study provides further evidence that functional coupling of the recombination machinery with the structural
components that organize the chromosomes during prophase I is
essential for the controlled formation of COs during meiosis in
plants, as in other organisms.
METHODS
Plant Material
Barley (Hordeum vulgare cv Golden Promise) was grown to maturity in
compost in a standard heated greenhouse at 22°C under 16-h photoperiod where supplementary lighting was provided by high-pressure
sodium vapor lamps (Powertone SON-T AGRO 400W; Philips Electronic
UK). Pollen mother cells were harvested from the inflorescences and leaf
samples taken for genomic DNA extraction.
Multiple Alignment and Phylogenetic Analysis
The plant genomes platform Plaza 2.5 (http://bioinformatics.psb.ugent.
be/plaza/; Van Bel et al., 2012) was searched with Arabidopsis thaliana
ZYP1a. The plant ZYP1 amino acid sequences were aligned using the
National Center for Biotechnology Information multiple protein alignment tool (http://www.ncbi.nlm.nih.gov/tools/cobalt/cobalt.cgi?link_
loc=BlastHomeAd), and the full-length sequences were selected to
9 of 12
construct the phylogenetic tree using Drosophila melanogaster sequence as the out-group. Selected protein sequences were aligned
again using MUSCLE 3.7 and the alignment refined using Gblocks
0.91b using the pipeline of software tools available within www.
phylogeny.fr (Dereeper et al., 2008). The parameters used for MUSCLE
3.7 were the default when configured for highest accuracy and for
Gblocks were: minimum length of a block after gap cleaning= 10; no
gap positions were allowed in the final alignment; all segments with
contiguous nonconserved positions bigger than eight were rejected;
and minimum number of sequences for a flank position = 85%.
Phylogeny was then constructed within the TOPALi interface (Milne
et al., 2009) with JTT+G as the substitution model. This model was
then used to estimate a Bayesian phylogenetic tree using MrBayes
v3.1.1 (Ronquist and Huelsenbeck, 2003) launched from TOPALi. The
Bayesian analysis settings were two runs of 625,000 generations and
a 20% burn-in with trees sampled every 10 generations. The posterior
probabilities higher than 70% show support for each cluster. The
alignments on which the phylogenetic tree is based are provided in
Supplemental Data Set 1.
Zyp1-RNAi Construct and Transgenic Lines
A 39-end fragment of 800 bp was amplified by PCR using two ZYP1-specific
Gateway primers attB1-HvZYP1 and attB2-HvZYP1 (Supplemental Table 3)
and the FL-HvZYP1 plasmid DNA as template. The obtained PCR fragment
was cloned into the entry vector pDONR207 using BP clonase II (Invitrogen).
The resulting entry clone was used to transfer the fragment into the RNAi
destination vector pBract207 (John Innes Centre) using LR Clonase (Invitrogen). The final construct where the RNAi cassette is under the transcriptional control of the constitutive ZmUbiquitin promoter was transferred
into Agrobacterium tumefaciens AGL1 containing the helper plasmid
pSOUP. Barley cv Golden Promise transformation was performed by the
FunGen Group at The James Hutton Institute using immature embryos
(Harwood et al., 2009).
Screening of Transgenic ZYP1-RNAi Lines
The T0 transgenic lines were grown to maturity and T1 seed number per
tiller scored. Transgene segregation rate was determined by the root
assay described by Jacobsen et al. (2006). Twenty seeds per line were
germinated on solid agar (0.5% phytogel) in the presence of 100 µg/mL of
hygromycin. The resistant transgenic seedlings and their segregating
sensitive azygotes were scored to estimate the number of the transgene
loci in each line. Based on preliminary DAPI staining of nuclei and preliminary fertility estimates from seed numbers, five semisterile lines (15,
24, 30, 35, and 43) were selected. For these, T1 seeds were germinated
without hygromycin selection and grown to maturity for further analysis.
The presence or absence of the transgene construct in these plants was
determined by PCR using their genomic DNA and hygromycin genespecific primers (Supplemental Table 3).
Determination of ZYP1 Expression Using qPCR
Three to five plants were grown for each selected RNAi line, empty vector
control, and untransformed barley Golden Promise cultivar, and three
samples per plant were collected for RNA extraction. Total RNA was extracted from leaf, root, stem, and young inflorescences (1.0 to 1.5 cm length)
using TRI reagent (Sigma-Aldrich), treated with RNase-Free DNase (catalog
number 79254; Qiagen), and then cleaned with RNeasy Mini Spin Columns
(catalog number 74104; Qiagen). Purified RNA (2 mg) was used to make the
first-strand cDNA using Superscript Vilo cDNA synthesis kit (Invitrogen).
qPCR was performed using the StepOnePlus Real-time PCR system
machine from Applied Biosystems and FastStart Universal Probe Master
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The Plant Cell
(ROX) from Roche. qPCR was performed using Hv-ZYP1–specific primers
Hv-ZYP1L and Hv-ZYP1R and their corresponding Roche Universal
ProbeLibrary probe number 65 (catalog number 04688643001). The
barley SKP1-specific (S-phase kinase-associated protein 1; accession
number AK249865) primers SKP1L and SKP1R were used for internal
reference in the presence of Roche Universal ProbeLibrary probe number
71 (catalog number 04688945001) (see Supplemental Table 3 for oligonucleotide list). The qPCR results were analyzed using StepOnePlus
software V2.2.2. ANOVA was performed using the log of the relative expression value plus one in order to ensure that model assumptions were
met. A significant difference [F(8,63) = 72.79, P < 0.001] was found between
lines. An LSD at the 5% level was used to compare ZYP1RNAi lines and their
corresponding azygotes to the empty vector control.
Cytological Procedures
PMCs were examined by light microscopy after staining with DAPI, and
chiasmata were recorded after FISH with 5S (pTa794) and 45S (pTa71)
probes to identify individual bivalents (Leitch and Heslop-Harrison, 1993;
Taketa et al., 1999; Sanchez-Moran et al., 2001). Immunolocalization and
other cytological techniques were performed according to Higgins (2013).
Antibodies were used at a concentration of 1:200 including At-ASY1 rat, AtZYP1 rat, gH2AX rabbit (Millipore), At-RAD51 rabbit, At-MSH4 rabbit, and
Hv-MLH3 rabbit (Phillips et al., 2013). For the cytological time course,
10 mM BrdU (0.5 mL) was injected into barley influorescences, which were
then fixed in 3:1 ethanol:acetic acid at various time points and detected with
BrdU labeling and detection kit I (Roche Diagnostics), as in Higgins (2013).
Supplemental Figure 5. The Numbers of Chiasmata in Individual
Chromosomes Are Differentially Affected in the ZYP1RNAi Lines.
Supplemental Figure 6. Immunolocalization of gH2AX in the Empty
Vector Control and ZYP1RNAi Lines.
Supplemental Figure 7. Immunolocalization of RAD51 in the Empty
Vector Control and Selected ZYP1RNAi Lines.
Supplemental Figure 8. Immunolocalization of MSH4 (Early Leptotene) in the Empty Vector Control and ZYP1RNAi Lines.
Supplemental Figure 9. Immunolocalization of MSH4 (Late Leptotene) in the Empty Vector Control and ZYP1RNAi Lines.
Supplemental Figure 10. Immunolocalization of MLH3 in the Empty
Vector Control and ZYP1RNAi Lines.
Supplemental Table 1. Segregation of Hygromycin Resistance in the
T1 Seed of ZYP1RNAi Lines and Empty Vector Control Line (EV) When
Seeds Were Germinated on Hygromycin-Containing Medium.
Supplemental Table 2. Quantification of Recombination Protein
Loading in the Empty Vector Control and ZYP1RNAi Lines.
Supplemental Table 3. Oligonucleotides Used in This Study.
Supplemental Data Set 1. Multiple Sequence Alignment on Which the
Phylogenetic Tree in Figure 1 Is Based.
ACKNOWLEDGMENTS
Microscopy
Fluorescence microscopy was performed using a Nikon Eclipse 90i microscope. NIS-elements software (Nikon) was used to record numbers of
foci and trace chromosomes and to determine nucleus size. Statistical
analysis was performed using Minitab and one-way ANOVA. Numbers
after 6 are SE.
This work was funded by Biotechnology and Biological Science Research Council Grants BB/F020872/1 and BB/F019351/1. I.C. was
funded by European Community Grant FP7 222883. We thank Miriam
Schreiber for help with phylogenetic analysis.
Accession Numbers
AUTHOR CONTRIBUTIONS
Sequence data from this article used for the phylogenetic tree in Figure 1
can be found in the GenBank/EMBL data libraries under the following
accession numbers: barley (AFP73614), D. melanogaster (NP_650490),
wheat (Triticum aestivum; AFD04131), Arabidopsis (AT1G22260 and
AT1G22275), Brachypodium distachyon (XP_003579875), Theobroma cacao (EOY16005), tomato (Solanum lycopersicum; XP_004238527.1), Ricinus communis (XP_002513917), grape (Vitus vinifera; XP_003634472.1),
Brassica oleracea (ABO69625.1), potato (Solanum tuberosum;
XP_006364852.1), rice (Oryza sativa; ADD69817.1), maize (Zea mays;
NP_001182397.1), and Setaria italica (XP_004975811.1). Sequence data
also are available at Ensembl: soybean (Glycine max; GLYMA13G01840
and GLYMA14G34810).
C.H., F.C.H.F., R.W., and S.J.A. designed the research. A.B., J.D.H., S.V.,
R.M.P., and L.R. performed the research. J.S. produced the transgenic
plants. H.O. performed statistical analysis. J.D.H., F.C.H.F., S.J.A., I.C.,
A.B., and C.H. wrote the article.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Determining the Number of T-DNA Inserts in
ZYP1RNAi Lines Using Fluorescence in Situ Hybridization with pBRACT
Probe on Meiotic Prophase I Nuclei.
Supplemental Figure 2. Hv-ZYP1 Suppression Induces Sterility in
Barley.
Supplemental Figure 3. Unmerged Images from Figure 4 of ZYP1
(Green) and ASY1 (Red) of Prophase I in Empty Vector Control Nuclei
and of Prophase I in ZYP1RNAi Nuclei.
Supplemental Figure 4. A Cytological Analysis of Meiotic Chromosomes in ZYP1RNAi Lines That Were Not Shown in Figure 5.
Received November 27, 2013; revised January 17, 2014; accepted
February 2, 2014; published February 21, 2014.
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The Synaptonemal Complex Protein ZYP1 Is Required for Imposition of Meiotic Crossovers in
Barley
Abdellah Barakate, James D. Higgins, Sebastian Vivera, Jennifer Stephens, Ruth M. Perry, Luke
Ramsay, Isabelle Colas, Helena Oakey, Robbie Waugh, F. Chris H. Franklin, Susan J. Armstrong and
Claire Halpin
Plant Cell; originally published online February 21, 2014;
DOI 10.1105/tpc.113.121269
This information is current as of June 16, 2017
Supplemental Data
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